ExoDNAPS™ Assay Protocol:
- Plasma and Serum sample preparation: Prepare plasma/serum samples by 3 centrifugation steps to eliminate red blood cells and cellular debris.
- 10 min at 300g (save supernatant, discard pellet)
- 20 min at 1200g (save supernatant, discard pellet)
- 30 min at 10,000g (save supernatant, discard pellet)
- Reagent preparation:
- Washing Buffer 1 and Washing buffer 2: Add the volume of pure ethanol (96 % ) indicated on the label of the bottles of both Washing Buffers.
- ExoPureTM, Elution buffer and Lysis buffer are ready to use.
- Lyophilized Exosome Standards:
- Reconstitute lyophilized exosome standard by adding 100 μL of deionized water and pipetting the solution up and down 10-15 times, avoiding bubbles. Vortex the reconstituted standard for 60 sec. Briefly centrifuge the tubes containing the standard to ensure that the solution is collected at the bottom of the tube.
- Exosome isolation (from Plasma and Serum):
- Add 125 μL of ExoPureTM reagent (ratio ExoPureTM/Sample 1/4) to 500 μL of precleared sample.
- Mix well by pipetting and inverting the tube.
- Incubate on ice for 1 hour.
- Centrifuge 20 min at 10,000g (centrifuge can be performed at 4 °C as well as at RT).
- Discard the supernatant.
- Eliminate the remaining supernatant into the tube with a tip.
- Resuspend isolated exosomes in 200 μL of 1x PBS.
- DNA Extraction:
- Add 20 μL of Proteinase K (600 mAU/mL).
- Add 200 μL of Lysis Buffer.
- Add 5 μL of Poly A Carrier (optional)*.
- Mix well by vortexing 30 sec.
- Incubate samples at 56 °C for 10 min. The use of Poly A Carrier results in an increase in DNA yield, in particular when small amounts of DNA are purified through silica membrane columns. No adverse effect on downstream applications such as PCR have been observed (Shaw et al. 2009).
- DNA Purification:
- Add 200 μL of Ethanol 96 % and mix by inverting the tube 6-8 times.
- Transfer the mixture in a Spin Column and centrifuge at 10,000 g for 1 min. Discard the flow-through.
- Add 500 μL of Washing Buffer 1, centrifuge for 1 min and discard the flow-through.
- Add 500 μL of Washing Buffer 2, centrifuge for 1 min and discard the flow-through.
- Centrifuge 2 additional min at 16,000g.
- Transfer the spin column to an Elution Tube.
- Elute the DNA from the column adding 50 μL of Elution Buffer.
- Incubate for 5 min at room temperature.
- Centrifuge 1 min at 200g.
- Centrifuge 1 min at 16,000g.
- Sensitivity: ExoDNAPSTM kit guarantees high efficiency isolation of circulating and Exosome-associated DNA.